Biological marker for stress states

ABSTRACT

The object of the invention is to propose a novel biomarker for stress states, which biomarker is highly meaningful and capable of enabling quantitative measurements to be made of stress levels. To do this, it has been found, surprisingly, that the air at the end of a person&#39;s breathing out, known as “alveolar” air, contains fluorine compounds when that person has been subjected to various stress states. More precisely, the present invention provides components based on fluorine compounds, in particular fluorinated chloroalkanes, used as biomarkers of stress states.

The present invention relates to a biological marker (or biomarker) thatreveals stress states, to a method of quantitatively evaluating thepresence of said biological marker of stress states, and to the use ofsuch a marker prepared in a packaged form for preventing stress states.

BACKGROUND OF THE INVENTION

The concept of stress states covers those metabolic and/or behavioralreactions that are provoked in an organism as a whole by numerousexogenous aggression factors such as: inflammatory illnesses; surgery;traumatic shocks; solar, electromagnetic, or ionizing radiation;smoking; pollution; allergies; prolonged effort; emotion; cold; etc.

The type of disturbance engendered in the organism enables differenttypes of states of stress to be distinguished, where stress can be of:chemical, microbiological, biochemical, physiological, psychic,biophysical, or pharmacological order.

It is well established that under the effect of some of theabove-mentioned factors, the organism produces oxidizing agents thatgenerate a kind of stress referred to as oxidation stress. These agentsare in the form of reactive oxygen species (ROS). For example, in theevent of prolonged effort, demand for oxygen increases, and consequentlyoxygen consumption increases, thereby leading both to a state of hypoxiaand to overproduction of ROS agents.

The production of ROS agents is then associated with normal endogenousbiochemical mechanisms. In particular, in the respiratory system, 2% to4% of the oxygen involved is reduced incompletely giving rise to ROSagents.

ROS agents are free radicals, atoms, or molecules that are unstable andreactive, usually presenting one or more lone electrons. The main ROSagents are: singlet oxygen (O—), superoxide anion, hydrogen peroxide,hydroxyl radical, nitrogen monoxide, or hydroperoxy radicals (producedduring peroxidation of membrane lipids, particularly those constitutedby polyunsaturated fatty acids).

Biologically speaking, oxidation stress leads to:

-   -   lipid peroxidation targeted on cell membranes and mitochondrial;        polyunsaturated fatty acids (PUFA) are attached and released        ethane (ω-3 PUFA) and pentane (ω-6 PUFA);    -   protein oxidation of mitochondrial proteins, leading to        malfunction of the respiratory system and to a reduction in the        amount of energy produced by cells; or    -   oxidation of mitochondrial DNA (mtDNA) which leads to mutations        that also lead to malfunction of the mitochondria.

Oxidation is the main cause of cellular aging and of diseases due to age(cancers, cardiovascular disorders, reduced immune functions, brainmalfunction such as Alzheimer's disease, or cataracts). This iscorroborated by the fact that anti-oxidant foodstuffs (ascorbic acid,tocopherol, and carotenoids of fruit and vegetables) contribute tocombatting the appearance of such degenerative diseases.

Ethane and pentane constitute known biomarkers for oxidation stressstates. As mentioned above, oxidation stress gives rise to metabolicdisorders including lipid peroxidation which leads to the formation ofethane and pentane. These products are volatile substances which aresubsequently eliminated in breathed-out air.

Pentane appears to be more significant than ethane in vivo since, inmembranes, lipids of ω-6 PUFA structure predominate over those of ω-3PUFA structure. The measured ethane/pentane concentration inbreathed-out air is proportional to the oxidation stress state.

Nevertheless, it is important to have a quantitative marker for stressstates, and in particular for oxidation stress.

OBJECT AND SUMMARY OF THE INVENTION

The object of the invention is thus to propose a novel biomarker ofstress states, which is highly significantly and which can enable stresslevels to be measured quantitatively.

To do this it has been found, surprisingly, that the air at the end of aperson's breathing out, referred to as “alveolar air”, contains fluorinecompounds if the person has been subjected to various stress states.

More precisely, the present invention provides a product of fluorinecompounds used as a biomarker of stress states, in particular compoundsbased on fluorinated chloroalkanes (also known as CFCs or freons).

More precisely, the product is obtained from a biological fluid, i.e. aliquid or gaseous material produced by the human body, by fractioningand concentrating on final portions of samples of said fluid. The fluidmay be constituted, for example, by air that has been breathed out, byblood, or by urine.

Preferably, the product of the invention is contained in a fraction ofalveolar air obtained by concentrating air breathed out by the humanbody. The biomarker is based on at least one of the components selectedfrom: trichloro-trifluro-ethane; tetrachloro-hexafluoro-butane; andtrichloro-monofluoro-methane. Since it is the most abundant, it ispreferable to select trichloro-trifluro-ethane.

The invention also provides a method of quantitatively evaluating thepresence of said marker, the method consisting in taking andconcentrating a sample of material, in analyzing the concentratedproduct by thermal desorption, gas chromatography coupled with a massspectrometer in order to identify the presence of CFCs, and inparticular of C₂Cl₃F₃, and then by calibration of the chromatographicpeak of the biomarker.

A quantitative determination of a stress level for each stress state canthen be performed by calibration, in direct association with thequantitative evaluation of the presence of the biomarker of theinvention. The result of such determination can serve as an intermediatebase for subsequent medical diagnostic purposes (inflammation, etc. . .. ).

The invention also relates to using fluorine compounds limiting fluorinelosses and thus losses of fluorine derivatives, prepared as medicationor as a food additive, to act as an agent for preventing stress, usingan appropriate formulation of the active principle. Such fluorinecompounds can be sodium fluorides (e.g. monofluorophosphate), CFCs,fluorine-containing amino acids and derivatives thereof (e.g. aminefluoride, etc.).

Such compounds can be administered under a variety of forms: tablets, anancillary compound for a medical device such as a patch or a dressing,or indeed as a liquid formulation additive in an ionophoretic device forpercutaneous administration.

BRIEF DESCRIPTION OF THE DRAWINGS

Other characteristics and advantages will appear on reading thefollowing detailed description relating to embodiments of the inventionfor different stress states given with reference to the accompanyingfigures, in which, respectively:

FIG. 1 shows two chromatograms of alveolar air sampled on a personrespectively at rest and after walking fast for 15 minutes;

FIG. 2 is a chromatogram of alveolar air sampled on a person 5 minutesafter intensive sports effort undertaken for 1 hour;

FIG. 3 is a chromatogram of alveolar air taken from the same person 6hours after the intense sports effort;

FIG. 4 is two chromatograms of alveolar air taken from an occasionalsmoker respectively at rest and 5 minutes after smoking; and

FIG. 5 is two superposed chromatograms of alveolar air takenrespectively from an occasional smoker 5 minutes after smoking and froma chronic smoker at rest.

MORE DETAILED DESCRIPTION

The device for sampling breathed-out air as used for performing themeasurements described below is of the type described in the patentapplication published under the No. FR 2 800 465, filed in the name ofthe Applicant and incorporated by reference. That device enables air atthe end of breathing out, known as “alveolar air” to be taken andconcentrated, which air contains a rich fraction of biomarkers presentin pulmonary air, is highly sensitive, and presents a high level ofreproducibility between samples. Common protocols relating to conditionsfor applying stress and for taking samples of air were complied with inthe various examples described below, in order to verify that theresults are reproducible.

The samples that were taken were in the form of cartridges filled withtwo layers of adsorbent material. The various samples were analyzed bythermal desorption, followed by gas chromatography with detection bymass spectrometry.

The chemical family of fluorinated chloroalkanes, also referred to aschloro-fluoro-carbons (CFCs) or freons, were thus measured as biomarkersfor stress in the breathed-out alveolar air. In the examples thatfollow, tri-chloro-trifluoro-ethane C₂Cl₃F₃ is the biomarker that showsup most particularly.

The chemical structure of this biomarker was determined by massspectrometry associated with gas chromatography.

The examples of increases in the production and in the elimination ofthe biomarker of the invention as illustrated below were caused byvarious different types of stress.

With reference to FIG. 1, moderate effort was used to illustratephysiological stress. The two chromatograms shown, C1 and C2, correspondto chromatographic analysis of the alveolar air sampled on the sameperson respectively at rest (curve C1) and after walking fast for 15minutes (fragmentary curve C2). The intensity I of the signal is plottedas a function of time t. The units are arbitrary. The two chromatogramsare slightly offset in order to improve the visibility of the figure.

The peaks P1 correspond to the presence of C₂Cl₃F₃, the peaks P2 to thepresence of isoprene, and the peaks P3 to the presence ofdichloromethane. The difference in intensity between the peaks P1 showsclearly that C₂Cl₃F₃ is sensitive as a biomarker for stress of thephysiological type.

FIGS. 2 and 3 relate to effort causing the person to be out of breath,caused by playing tennis for 1 hour, and is likewise for the purpose ofillustrating physiological stress.

In FIG. 2, the chromatogram C3 of alveolar air was taken from the person5 minutes after the sports effort. The high amplitude of the peak P1represents the level of stress to which the organism of the player wassubjected. The peaks P4 to P12 correspond respectively to cyclohexane(P4), to hexane (P5), to chloroform (P6), to methylcyclohexane (P7), totrimethylhexane (P8), to heptane (P9), to tetrachloroethylene (P10), tobenzene (P11), and to ethylether (P12).

In FIG. 3, i.e. 6 hours after the intensive sports effort, the peak P1of the chromatogram C4 has diminished significantly, thereby furtherillustrating the dedicated stress biomarker function associated withtrichlorotri-fluoroethane C₂Cl₃F₃.

FIGS. 4 and 5 show that C₂Cl₃F₃ is also sensitive in revealing chemicaltype stress.

In FIG. 4, two chromatograms C5 (slightly offset) and C6 represent thealveolar air taken from an occasional smoker respectively at rest and 5minutes after smoking.

The peaks P1 correspond again to the presence of C₂Cl₃F₃, the peaks P2to the presence of isoprene, and the peaks P3 to the presence ofdichloromethane. Other peaks are identified: the peak P6 corresponds tochloroform and the peak P12 to ethylether.

In FIG. 5, the two chromatograms comprising superposed curves C6 and C7represent alveolar air taken respectively from the occasional smoker, 5minutes after smoking as in the preceding figure, and for comparisonfrom a chronic smoker at rest, who had not smoked for at least 1 hourprior to the sample being taken. In this Figure, the hexane peak P5 andthe chloroform peak P6 of heptane are also shown.

A comparison between the peaks P1 in the two chromograms shows clearlythat the biomarker of the invention, C₂Cl₃F₃, is a good marker of atemporary stress state but not of a chronic state. The quantity ofC₂Cl₃F₃ was calibrated relative to dichloromethane which is present inalveolar air and which varies little, the calibration being in the formof the following ratio F between the intensities of the signals for thechromatograph peaks:$F = \frac{{chromatographic}\quad{peak}\quad{signal}\quad{for}\quad{biomarker}}{{chromatographic}\quad{peak}\quad{signal}\quad{for}\quad{dichloromethane}}$

Such calibration F is summarized in the table below for various examplesof stress states taken from a rest state, and in general 5 minutes afterthe end of various causes of stress (unless stated to the contrary), andspecifically: physiological stress due to practicing sports (violent andmoderate), biochemical stress due to smoking, psychic stress due toplaying a fast video game intensely, and biophysical stress due toexposure to pulsed electromagnetic radiation from a computer screen.

The calibrated measurements F of the biomarker taken at rest (F₀) andafter being subjected to various kinds of stress in the conditionsstated, are summarized in the following table: Type of stress F₀ Howapplied F Moderate 0.37 20 minutes exercise bicycle 0.48 physicalModerate 0.13 15 minutes walking fast 3.14 physical Violent 0.21 1 hourof tennis 7.87 physical 6 hours after the effort 0.59 Biochemical 0.13Smoking a cigarette 12.5 Psychic 0.37 30 minutes of intense playing 5.00of a fast video game Biophysical 0.21 30 minutes exposure to 0.88electromagnetic radiation from a computer screen 15 minutes after theexposure 0.44 90 minutes after the exposure 0.21

Thus, whatever the type of stress, it is possible from the valuesobtained to quantify stress levels for each type of stress.

The invention is not limited to the examples described and shown. Forexample, the fluorine compound products may be used not only forprophylactic purposes but also for stress-curing purposes, at adetermined dosage. Furthermore, the use of fluorine compounds as apreventative agent can be extended to endogenous fluorine compounds andto compounds suitable for mobilizing them.

1. A product of compounds based on fluorinated chloroalkanes used as abiomarker for stress states.
 2. A product based on fluorinatedchloroalkanes according to claim 1, the product being obtained from abiological fluid produced by the human body, by fractioning andconcentrating final portions of samples of said material.
 3. A productbased on fluorinated chloroalkanes according to claim 2, the productbeing contained in a fraction of alveolar air obtained by concentratingair breathed out by the human body.
 4. A product according to claim 1,the product being based on at least one of the components selected from:trichloro-trifluro-ethane, tetrachloro-hexafluoro-butane, andtrichloro-monofluoro-methane.
 5. A method of quantitatively evaluatingthe presence of the product according to claim 1, as a biomarker ofstress states, the method consisting in taking and concentrating asample of material, in analyzing the concentrated product by thermaldesorption, gas chromatography coupled with mass spectrometry, in orderto identify the presence of CFCs, and then in calibrating thechromatographic peak of the biomarker.
 6. An evaluation method accordingto claim 5, in which quantitative determination of a stress level foreach stress state is subsequently performed by calibration.
 7. The useof compounds based on fluorine salts, on fluorine-containing aminoacids, and/or on fluorinated chloroalkanes in preparing medication or afood additive as an agent for preventing stress.
 8. Conditioningfluorine compounds for the use according to claim 7, the conditioningbeing selected from a tablet, an ancillary compound for a medicaldevice, and a liquid formulation additive in an ionophoretic device forpercutaneous administration.